The complement system is a set of 40-50 proteins in the blood that provides a first line of defence against infection. An improperly regulated complement system can damage host or self-cells as well as bacterial ones. It can thus cause or exacerbate the symptoms of many diseases. There is consequently much interest in developing reagents that monitor or modulate the proteins of the complement system.
Human complement factor H (CFH) is a protein that acts selectively on self-surfaces in order to protect them from complement system-mediated damage. Yet many pathogens can exploit host CFH for their own purposes. Studying how CFH (consisting of 20 complement control protein (CCP) modules, also referred to as short consensus repeats (SCRs) or sushi domains) interacts with the relevant microbial proteins has provided clues as to how CFH can be captured and its activity modulated by polypeptides produced in the laboratory.
PspC is one of four designations for a pneumococcal surface protein whose gene is present in approximately 75% of all Streptococcus pneumoniae strains. Under the name SpsA, the protein has been shown to bind secretary immunoglobulin A (S. Hammerschmidt, S. R. Talay, P. Brandtzaeg, and G. S. Chhatwal, Mol. Microbiol. 25:1113-1124, 1997). Under the name CbpA, the protein has been shown to interact with human epithelial and endothelial cells (C. Rosenow at al., Mol. Microbiol. 25:819-829, 1997). The gene is paralogous to the PspA gene in S. pneumoniae and was thus called PspC (A. Brooks-Walter, R. C. Tart, D. E. Briles, and S. K. Hollingshead, Abstracts of the 97th General Meeting of the American Society for Microbiology 1997). Under the name HIC, the protein has been shown to interact with human factor H (Janulczyk R., Iannelli P., Sjoholm A. G., Pozzi G., Bjorck L. J. Biol. Chem. 275:37257-37263, 2000). A detailed description of PspC is provided in Brooks-Walter et al., “The pspC Gene of Streptococcus pneumoniae Encodes a Polymorphic Protein, PspC, Which Elicits Cross-Reactive Antibodies to PspA and Provides Immunity to Pneumococcal Bacteremia” Infect Immun. 1999 December; 67(12): 6533-6542. PMCID: PMC97064. In this paper sequence comparisons of five published and seven new alleles reveal that this gene has a mosaic structure, and modular domains have contributed to gene diversity during evolution. Two major clades exist: clade A alleles are larger and contain an extra module that is shared with many PspA alleles; clade B alleles are smaller and lack this PspA-like domain. All alleles have a proline-rich domain and a choline-binding repeat domain that show 0% divergence from similar domains in the PspA protein.
The present inventors produced a Streptococcus pneumoniae PspC truncation (PspCN) that binds CFH irreversibly (on the biological timescale) primarily via interactions with CCPs 8-10. PspCN-captured CFH adopts a conformation in which a chemical cross-linker can conjoin its CCP 5 and CCP 18, as well as its CCP 7 and CCP 20; it is of interest that CCPs 7 and 20 mediate self-surface recognition and are dual binding sites for numerous other CFH-interacting bacteria.
In experiments conducted using surface plasmon resonance, the PspCN:CFH complex was found to bind at least threefold better than CFH to immobilised C3b, the activated version of complement component C3. The PspCN:CFH complex bound many times better than CFH to the proteolytic C3b fragment, C3d. The PspCN:CFH complex dissociated the C3 convertase, C3b.Bb (the enzyme that cleaves C3 to C3a and C3b) up to tenfold more effectively than CFH. These observations were speculatively attributed to exposure upon complex formation of an occluded surface-interacting and C3d/C3b-binding site within CCPs 19-20 of CFH.
In support of this notion, it was found that CFH (D1119G)—which lacks a critical residue within the C3d/C3b:CCP 19 interface and is linked to the kidney disease atypical haemolytic uraemic syndrome—bound to C3b immobilised on a surface plasmon resonance chip, and decayed similarly immobilised C3b.Bb, just as effectively as CFH (wild-type): yet this mutant did not prevent complement-mediated haemolysis of sialic acid-bearing erythrocytes, implying that CCPs 19-20 are utilised for the regulatory action of CFH on self-surfaces even though they do not contribute to binding C3b on a surface plasmon resonance chip.
Importantly, PspCN:CFH (D1119G) bound many fold more weakly than PspCN:CFH (wild-type) to C3d and showed little enhancement, over CFH (D1119G) alone, of C3b.Bb-decay acceleration. Hence, it was hypothesised by the inventors that CFH “hides” regulatory sites to avoid bacterial exploitation but PspC exposes them. Similar FH conformational switches may operate on self-surfaces.
There is an unmet need for methods and compositions for selectively binding complement components, in particular CFH, to solid supports and for modulating complement activity.
The ability to modulate complement activity opens up opportunities, inter alia, for treating diseases associated with aberrant complement activity.